Molecular Characterization of Human Skin Response to Diphencyprone at Peak and Resolution Phases: Therapeutic Insights  Nicholas Gulati, Mayte Suárez-Fariñas, Judilyn Fuentes-Duculan, Patricia Gilleaudeau, Mary Sullivan-Whalen, Joel Correa da Rosa, Inna Cueto, Hiroshi Mitsui, James G. Krueger  Journal of Investigative Dermatology  Volume 134, Issue 10, Pages 2531-2540 (October 2014) DOI: 10.1038/jid.2014.196 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Visualization of inflammatory reactions induced by diphencyprone (DPCP) over time. Clinical photography with ultrasound imaging below of a skin site (a) treated with placebo, (b) 3 days after treatment with DPCP, (c) 7 days after treatment with DPCP, and (d) 14 days after treatment with DPCP. Brackets on ultrasound images indicate extents of inflammatory reactions as reflected by dermal thickness. Shown is a representative subject (subject 008). Journal of Investigative Dermatology 2014 134, 2531-2540DOI: (10.1038/jid.2014.196) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Reactions to diphencyprone (DPCP) include immune activation markers that are present at day 3 but diminish over time. (a, b) Immunohistochemistry for (a) CD3 and (b) CD8 on placebo-treated samples, DPCP day 3 samples, DPCP day 14 samples, and DPCP late samples. Scale bar=100 μm. (c) Reverse-transcriptase–PCR (RT–PCR) analysis for IFNγ, IL-2, IL-2RA, IL-13, IL-9, IL-17A, IL-22, and forkhead box P3 (Foxp3). Shown are average normalized expression values for placebo-treated day 3 samples (light blue bars, n=11), placebo-treated day 14 samples (dark blue bars, n=11), DPCP day 3 samples (pink bars, n=11), DPCP day 14 samples (red bars, n=11), and DPCP-treated samples 4–8 months after challenge (late) (brown bars, n=6). *P<0.01 when compared with placebo and error bars represent SEM. (d) Principal component analysis (PCA) of microarray data showing all samples as individual dots colored according to sample type. The larger dots are averages for each sample type. Journal of Investigative Dermatology 2014 134, 2531-2540DOI: (10.1038/jid.2014.196) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Reverse-transcriptase–PCR (RT–PCR) and histological analysis of subjects whose CD3+ or CD11c+ infiltrates increase from 3 days to 14 days after diphencyprone (DPCP) challenge (subgroup A, n=5). (a) RT–PCR analysis for IFNγ (left panel), IL-2 (middle panel), and IL-2RA (right panel). Shown are normalized expression values for each subject individually to highlight that almost all samples have decreased expression of these genes at day 14 compared with day 3. (b–f) Hematoxylin and eosin (H&E; b) and immunohistochemical analysis of samples for (c) CD3, (d) CD11c, (e) lysosome-associated membrane glycoprotein, dendritic cell-specific (DC-LAMP), and (f) Langerin. For all histological images, left panels show placebo reactions, middle panels show DPCP day 3 reactions, and right panels show DPCP day 14 reactions. Shown is a representative subject (subject 013). For immunohistochemical stains, line graphs indicate subgroup-wide average cell counts for placebo, DPCP day 3, DPCP day 14, and DPCP late samples. *P<0.05 when compared with placebo. Scale bar=100 μm. Journal of Investigative Dermatology 2014 134, 2531-2540DOI: (10.1038/jid.2014.196) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Negative regulatory immune cells and molecules are increased in diphencyprone (DPCP) day 3 reactions of subgroup B compared with subgroup A. (a, b) Forkhead box P3 (Foxp3; red)–CD3 (green) immunfluorescence for (a) a representative subgroup A member (subject 012) and (b) a representative subgroup B member (subject 021). Left panels are DPCP day 3 samples and right panels are DPCP day 14 samples. Regulatory T cells (Tregs) were quantified as double positive for Foxp3 and CD3. Subject 012 had 4 double-positive cells at day 3 (subgroup-wide average of 9) and 24 at day 14 (subgroup-wide average of 20.33), whereas subject 021 had 37 at day 3 (subgroup-wide average of 21.25) and 19 at day 14 (subgroup-wide average of 12). (c–i) Reverse-transcriptase–PCR (RT–PCR) analysis for (c) IL-10, (d) cytotoxic T-lymphocyte antigen 4 (CTLA4), (e) programmed cell death 1 (PD1), (f) programmed cell death ligand 1 (PDL1), (g) programmed cell death ligand 2 (PDL2), (h) IDO1, and (i) lymphocyte activation gene 3 (LAG3). Shown are average normalized expression values of placebo day 3, placebo day 14, DPCP day 3, and DPCP day 14 skin for both subgroups (n=5 for subgroup A (blue bars), n=6 for subgroup B (green bars)). P-values are shown to compare subgroup A with subgroup B for each sample type. Scale bar=100 μm. Journal of Investigative Dermatology 2014 134, 2531-2540DOI: (10.1038/jid.2014.196) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions